1. Field of the Invention
This invention relates to recombinant DNA technology and more particularly to a new method for enhancing the production of heterologous proteins in bacterial host cells. The disclosed method involves infecting host cells, which contain plasmid encoding the gene of interest, with bacteriophage xcex to induce lysis of the bacterial host cells. Super-production may be achieved in selected host cells either when the plasmid alone carries at least one copy of the heterologous DNA or when both plasmid and phage xcex each carry at least one copy of the heterologous DNA.
2. Description of the Related Art
At present, genetic engineering methods allow creating microorganism strains capable of producing substantial amounts of various bioactive substances having important applications in medicine and industry. Typically, plasmid vectors into which a heterologous gene has been inserted are used to transform bacterial host cells. Different strains of E. coli are frequently used as recipient cells. Using such plasmid-dependent transformation methods, E. coli cells have been engineered to produce a variety of valuable human peptides and proteins, including insulin, xcex3-interferon, a number of interleukins, superoxidedismutase, plasminogen activator, tumor necrosis factor, erythropoietin, etc. These substances are either already used in medical practice or undergoing different stages of clinical studies.
However, the plasmid method has serious disadvantages. It is technologically complicated, since the desired product has to be extracted from bacterial cells after biosynthesis, which is a multi-stage process. For example, interferon extraction involves disintegration of cells, buffer extraction, polyethylene-imine processing, clarification, precipitation by ammonium sulfate, dialysis, and centrifugation (Goeddel, EP 0043980). The necessity for such extraction and purification steps not only complicates production technology of the recombinant product, but also results in substantial losses, especially during large-scale industrial production.
A further complicating factor is that at relatively high levels of expression of the cloned genes, the eukaryotic proteins generated tend to accumulate in the cytoplasm of E. coli as insoluble aggregates, which are often associated with cell membranes. Consequently, the already difficult extraction and purification methods discussed above must be supplemented with additional technical procedures related to the extraction of the insoluble inclusion bodies. Usually, the insoluble proteins are solubilized using ionic detergents, such as SDS or laurylsarcosine, at increased temperatures or in the presence of denaturants, such as 8 M urea or 6-8 M guanidine-HCl.
Often, the final stage of purification involves renaturation and reoxidation of the solubilized polypeptides, which is required to restore functional activity. Disulfide bonds, which are necessary for proper folding of the protein in its native conformation, must be reformed. Renaturation procedures, such as disulfide interchange, may use expensive and relatively toxic reagents, like glutathione, and oxidized 2-mercaptoethanol or dithiothreitol. Further, the final yield of bioactive genetically-engineered proteins may be relatively low. Moreover, the presence of even trace concentrations of the toxic reagents needed to solubilize and then re-establish secondary and tertiary protein structure may prohibit subsequent clinical application of the proteins. Thus, the generation of targeted protein in the form of insoluble inclusion bodies within the bacterial host cells not only complicates the production of recombinant proteins and results in diminished yield, but may also render the final protein unsuitable for clinical use (Fisher, B., Sumner, I., Goodenough, P. Biotech. and Bioeng. 41:3-13, 1993).
The technological difficulties associated with the extraction of proteins produced by the expression of heterologous genes from plasmid-transformed bacterial host cells may be overcome by infecting the transformed bacterial host cells with bacteriophage, whose lytic pathway results in lysis of the bearer cell. Thus, the desired protein may be simply released into the culture medium (Breeze A. S. GB 2 143 238A). Accordingly, Breeze disclosed a method of increasing the yield of enzyme produced in E. coli by infecting the bacterial cells with phage xcex carrying a temperature-sensitive mutation in cI to provide controlled lysis. The cI-gene product is a repressor of early transcription and consequently blocks transcription of the late region of the phage DNA, which is required for head and tail assembly and cell lysis (Mantiatis, T., Fritsch, E. F., Sambrook, J., MOLECULAR CLONING: A LABORATORY MANUAL, 1982, Cold Spring Harbor Laboratory Press). Bacteriophages carrying a temperature-sensitive mutation in cI are able to establish and maintain the lysogenic state as long as the cells are propagated at a temperature that allows the cI-gene product to repress transcription of phage genes necessary for lytic growth. Accordingly, the transformed bacterial host cells may be cultivated at 30xc2x0 C., wherein the cI-mediated suppression of phage DNA transcription continues and the phage remains in the lysogenic state, until the stage of maximum ferment production is reached. Subsequently, the culture temperature may be increased to 42xc2x0 C. for 30 minutes in order to inactivate the cI repressor and permit the phage to begin its lytic development. The host cells may then be incubated for 2-3 hours at 30xc2x0 C. to allow complete lysis and release of the enzyme (Breeze A. S. GB 2 143 238A).
Although Breeze teaches release of proteins from bacterial producer cells, it requires cultivating producers at temperatures not exceeding 30xc2x0 C., which is not the optimum temperature for growth of E. coli cells. Synthesis at the optimum temperature (37xc2x0 C.) is not possible, since cells at temperatures exceeding 32xc2x0 C. undergo lysis before reaching the stage of maximum ferment accumulation due to the development of temperature-sensitive lytic prophage. Furthermore, incubation of the bacterial host cells at 42xc2x0 C. for 30 min as disclosed by Breeze may activate proteases that destroy the targeted protein.
Auerbach et al. (U.S. Pat. No. 4,637,980) used a phage xcex DNA fragment for inducing lytic release of recombinant products. In that method, like Breeze, the temperature-sensitive mutation in xcex cI-gene product was used to provide temperature-dependent lysis of the bacterial host cells. The xcex DNA fragment in Auerbach maintained functional endolysin-encoding genes, N, Q, R and S, for producing lysozyme following inactivation of the cI repressor at 42xc2x0 C. Most of the remaining phage genes were deleted; mutations in O and P genes prevented replication of the phage DNA. Consequently, the xcex DNA was not a fully functional phage, capable of modulating expression of the targeted gene. Moreover, the xcex DNA of Auerbach was not suitable for use as a vector for carrying targeted genes. Further, as discussed above, incubation of the bacterial host cells at 42xc2x0 to 44xc2x0 C. for 90-120 min as disclosed by Auerbach may activate proteases that destroy the targeted protein.
In addition to providing for the lytic release of intact protein from bacterial producer cells, bacteriophages have also been used as an alternative to bacterial plasmid-based vectors, for carrying heterologous DNA into host bacterial cells. (Murray, N. E. and Murray, K., Nature 251:476-481, 1974; Moir, A., Brammar, W. J., Molec. gen. Genet. 149:87-99, 1976). Typically, amplification of genes and their products is achieved during lytic growth of the phage, wherein the phage genome is integrated into the bacterial host DNA (Panasenko, S. M., Cameron, J. R., Davis, R. V., Lehman, L. R., Science 196:188-189, 1977; Murray, N. E. and Kelley, W. S., Molec. gen. Genet. 175:77-87, 1979; Walter, F., Siegel, M., Malke, H., 1990, DD 276,694; Mory, Y., Revel, M., Chen, L., Sheldon, I. F., Yuti-Chernajovsky, 1983, GB 2,103,222A). Usually, either lysogenic cultures of recombinant phage xcex are used for infecting the bacterial host cells, or xe2x80x9cwarmed upxe2x80x9d bacterial cultures, already harboring recombinant lysogenic phage xcex, are grown up to amplify expression of the heterologous genes.
Although there are examples of the successful use of xcex vectors for expression of heterologous genes, xcex vectors have been used primarily for gene cloning. Once cloned, the genes are transferred to plasmid vectors for more effective expression. For example, when E. coli is infected by phage xcex Charon 4A C15, containing the human xcex2-interferon gene, the quantity of interferon in cell lysate constituted 7-8xc3x97106 units/liter. After the DNA fragment bearing targeted gene was recloned from phage to plasmid, xcex2-interferon yield increased to 1xc3x97108 units/liter (Moir, A., Brammar, W. J., Molec. gen. Genet. 149:87-99, 1976).
To increase the yield of heterologous protein generated in bacterial host cells by recombinant xcex vectors, mutations in the phage genome have been introduced that cause phage xcex to lose its ability to initiate bacterial cell lysis. Enhanced yield is thereby achieved by extending the period of time during which the heterologous gene is expressed by the bacterial host cells. Thus, for example, the yield of DNA ligase 1 in lysogenic cultures containing xcex gt4ligS prophage, with amber-mutation in the S gene, was five times greater than the yield of DNA ligase 1 in lysogenic cultures containing xcex gt4lig prophage without the amber-mutation (Panasenko, S. M., Cameron, J. R., Davis, R. V., Lehman, L. R., Science 196:188-189, 1977). The phage xcex S protein is required for lysis; therefore Sxe2x88x92 mutants accumulate large numbers of intracellular progeny phage particles, as well as the targeted protein, without lysing the host cells (Mantiatis, T., Fritsch, E. F., Sambrook, J., MOLECULAR CLONING: A LABORATORY MANUAL, 1982, Cold Spring Harbor Laboratory Press).
Similar increases in the yield of DNA polymerase 1 were reported for lysogenic cultures containing recombinant phage xcex with amber-mutations in the S and Q genes, compared to recombinant phage xcex without the amber-mutations (Murray, N. E. and Kelley, W. S., Molec. gen. Genet. 175:77-87, 1979). The phage xcex Q protein is required for transcription of the late region of the phage DNA, which includes many genes involved in head and tail assembly and cell lysis. (Mantiatis, T., Fritsch, E. F., Sambrook, J., MOLECULAR CLONING: A LABORATORY MANUAL, 1982, Cold Spring Harbor Laboratory Press).
In U.S. Pat. No. 4,710,463, Murray discloses lysogenizing a non-suppressing (Suxc2x0) strain of E. coli with phage xcex containing the temperature-sensitive mutation in cI, as well as mutations in xcex S and E genes. Consequently, prolonged cultivation of the lysogenic E. coli at 37xc2x0 C. leads to high levels of production of the recombinant protein, which is retained within the cells, since these are not lysed by phage gene products in the normal way, and since the recombinant phage genome is not encapisdated, it remains available for transcription.
Despite the enhanced yield of heterologous proteins possible using xcex-vectors with S and E mutations, the potential technical advantages of bacteriophage vectors related to the lytic release of targeted proteins, may be lost with these mutations, because the targeted proteins accumulate inside the bacterial cell. Thus, when a lysis-defective mutant xcex-vector is used for production of heterologous protein, the extraction and purification steps, discussed above for bacterial cells transformed by plasmid vectors, along with the resultant losses, must be performed.
The present invention discloses a method for producing a biologically active protein of interest. The method comprises the steps of: (1) transforming a bacterial host cell with a plasmid having at least one copy of an expressible gene encoding the protein, (2) infecting the transformed bacterial host cell with a bacteriophage capable of mediating lysis and also capable of lytic growth without lysis, and (3) cultivating the bacterial host cell under a culture condition that induces lytic growth of the cell without lysis until a desired level of production of the protein is reached.
In a preferred embodiment, the bacteriophage has a temperature-sensitive mutation. More preferably, the bacteriophage is bacteriophage xcex and the temperature-sensitive mutation is cI857. The culture condition that induces lytic growth of the bacteriophage is at a temperature of greater than 32xc2x0 C. Prior to the cultivating step, the bacterial host cells may be grown at a temperature, generally less than about 32xc2x0 C. that prevents lytic growth of the bacteriophage.
In a variation of the disclosed method, the bacteriophage has a mutation in at least one gene involved in bacteriophage-mediated lysis of the bacterial host cell. Preferably, the bacteriophage is bacteriophage xcex and the at least one gene involved in bacteriophage-mediated lysis is selected from the group consisting of N, Q and R. Moreover, the bacterial host cell is preferably from a strain of E. coli. The strain of E. coli may or may not produce a suppressor for the repair of amber-mutations.
Bacteriophage-mediated lysis of the bacterial host cell may be delayed by culturing at higher multiplicities of infection compared to lower multiplicities of infection. The infecting bacteriophage may be provided at a multiplicity of infection in a range of about 1 to about 100 and more preferably, at a multiplicity of infection in a range of about 10 to about 25.
In another aspect of the present invention, the bacteriophage may contain at least one copy of an expressible gene encoding the same heterologous protein which is encoded by the plasmid.
A variation of the method for producing a biologically active protein in accordance with the present invention is disclosed. The method comprises the steps of: (1) transforming a bacterial host cell with a plasmid having at least one copy of an expressible gene encoding the protein, (2) infecting the transformed bacterial host cell with a bacteriophage having at least one copy of an expressible gene encoding the protein, and (3) cultivating the bacterial host cell under a culture condition that allows expression of the plasmid and phage genes.
In accordance with another aspect of the present invention, a bacterial host cell is disclosed. The bacterial host cell has a plasmid with at least one copy of an expressible heterologous gene encoding a protein, wherein the host cell is infected with a bacteriophage capable of mediating lysis and also capable of lytic growth without lysis.
The bacterial host cell preferably has a bacteriophage with a temperature-sensitive mutation. More preferably, the bacterial host cell is infected with bacteriophage xcex and the temperature-sensitive mutation is cI857.
In a variation of the bacterial host cell, the bacteriophage has a mutation in at least one gene involved in bacteriophage-mediated lysis of the host cell. Preferably, the bacterial host cell is infected with bacteriophage xcex having a mutation in at least one gene selected from the group consisting of N, Q and R. More preferably, the bacterial host cell is infected with bacteriophage xcex with cI857, Qam117 and Ram54 mutations.
In a preferred embodiment of the bacterial host cell of the present invention, the host cell has a plasmid encoding a protein of interest and is also infected with a bacteriophage having at least one copy of an expressible gene encoding the protein of interest.
The bacterial host cell in accordance with the present invention is preferably a strain of E. coli. The strain of E. coli may or may not have a suppressor for repairing amber-mutations. Similarly, the strain of E. coli may or may not be recA deficient. One preferred strain of E. coli host cells in accordance with the present invention contains a plasmid having at least one copy of an expressible heterologous gene encoding a protein, wherein the strain of E. coli is infected with bacteriophage xcex having cI857, Qam117 and Ram54 mutations. The protein may be human alpha-2b interferon. More preferably, in addition to having a plasmid with at least one copy of a gene encoding a protein, the E. coli host cell also has a bacteriophage xcex having cI857, Qam117 and Ram54 mutations and at least one copy of a gene encoding the protein. This bacteriophage preferably lacks a suppressor for repairing amber-mutations.